Mol. Cells 2019; 42(2): 183-188
Published online January 24, 2019
https://doi.org/10.14348/molcells.2018.0382
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: hjoonkim@pusan.ac.kr
Osteoarthritis (OA) is a naturally occurring, irreversible disorder and a major health burden. The disease is multifactorial, involving both physiological and mechanical processes, but calcium crystals have been associated intimately with its pathogenesis. This study tested the hypothesis that these crystals have a detrimental effect on the differentiation of osteoclasts and bone homeostasis. This study employed an osteoblast-osteoclast coculture system that resembles
Keywords DUSP1, osteoarthritis, osteoclastogenesis, RANKL
Osteoarthritis (OA) is the most common joint disorder in humans (Fuerst et al., 2009). The disease is slowly progressive and involves all components of the joint, including the bone, cartilage, meniscus, and synovium (Macmullan and McCarthy, 2010). The high prevalence and related physical impairment make OA a leading cause of disability worldwide (McCarthy et al., 2009). Thus far, multiple predisposing factors have been identified, including genetic predisposition, joint trauma, obesity, sex, and hormonal status (Brandt et al., 1998). However, our understanding of the pathogenesis of the condition is limited and there is no pharmacological therapy yet to reverse or retard the consequences of OA (Qvist et al., 2008).
Calcification of the articular cartilage is a well-recognized feature of OA and current evidence suggests that it contributes directly to joint degeneration (MacMullan et al., 2010). Calcium pyrophosphate dehydrate (CPP) and basic calcium phosphate (BCP) are the two most common forms of calcium crystals found in the articular cartilage (Molloy and McCarthy, 2006). The presence of these crystals is associated with a number of clinical manifestations (MacMullan et al., 2010). Both types of crystals are found in OA. Individual crystals are typically less than 1 μm in size and they aggregate in the OA synovial fluid to form clumps that are approximately 5 to 20 μm (McCarthy et al., 2011).
In the present study, we hypothesized that these crystals may have a detrimental effect on the differentiation and survival of osteoblasts and osteoclasts. Recent studies have frequently used the term “coupling of bone and cartilage” to describe the crosstalk between the articular cartilage and subchondral bone in the synovial joints through increased vascularization and development of microcracks in the bone matrix (Burr and Radin, 2003; Lajeunesse and Reboul, 2003) and the cellular and molecular interactions between osteoblasts and chondrocytes that affects the initiation and progression of OA (Karsdal et al., 2008; Mansell et al., 2007; Sharma et al., 2013). Thus far, the pathogenic role of crystals in the degradation of the extracellular matrix and subchondral bone remodeling have been studied (Corr et al., 2017) but only few studies have examined their effects on osteoclasts and bone remodeling (Stack and McCarthy, 2016).
To test this hypothesis, amorphous calcium phosphate (ACP) arrays were first prepared on a 60 mm culture dish, as described previously (Kim et al., 2010). ACP arrays were fabricated on the surface of the dish to mimic the crystals observed in OA patients. As illustrated in Fig. 1(A), murine bone marrow-derived macrophages (BMMs) were cocultured with murine calvarial osteoblasts, as described by Ryu et al. (Ryu et al., 2006) either with or without a Ca2+-phosphate coating. Early studies of cocultures found that a direct cell-to-cell interaction was required for osteoclast generation, while the following studies showed that RANKL expressed on osteoblasts and RANK present on the osteoclast precursors were responsible for the cell surface molecular interaction for osteoclastic differentiation (Suda et al., 1999). The cocultures were treated with prostaglandin E2 (10−6 M) and vitamin D3 (10−8 M) to stimulate osteoblast differentiation and induce cell surface RANKL expression to facilitate the osteoclastic differentiation of BMMs, which resemble the actual osteoblast-dependent osteoclast differentiation
In addition, gene expression of the common markers of osteoclast activity, cathepsin K, and matrix metallopeptidase 9 (MMP9) was assessed (Fig. 1C). In keeping with the earlier results, it was found that the expression of those two genes were increased notably in osteoclastic cells cultured in the coated dish on day 6. In an attempt to identify the molecular mechanisms underlying the augmentation of osteoclastogenesis by the crystals in the coculture, the altered expression of genes between the non-coated and coated conditions were analyzed using MouseWG-6 v2.0 Expression BeadChip (Illumina). Microarray results from Fig. 1D revealed that the gene expression of some common markers of osteoblastic activity, such as collagen type 1, bone sialoprotein, and Runx2, did not show a significant difference between the two dishes on day 6 of coculture. Conversely, gene expression of RANKL, which plays an essential role in the commitment of precursors to osteoclastic differentiation (Boyle et al., 2003; Suda et al., 1999; Teitelbaum and Ross, 2003), was upregulated by 140% from 237.1 to 326.9, suggesting that a Ca2+-phosphate coating does not significantly alter osteoblast differentiation but enhances osteoclast differentiation.
The microarray results from cultures of osteoblasts grown on either non-coated or coated dishes for six days were next examined to determine the gene responsible for the increase in RANKL expression. Microarray analysis identified the genes that showed a greater than 1.35-fold difference in expression between the two dishes. Among the 167 genes, two genes reported the greatest difference and dual-specificity phosphatases 1 (DUSP1) was selected as the most relevant after pathway analysis using PANTHER (Mi et al., 2013). DUSPs are cysteine-based enzymes that can remove phosphate groups from phosphor-serine/threonine residues (Patterson et al., 2009) and play important roles in MAPK signaling pathway in the development and immune response (Nunes-Xavier et al., 2011). Among them, DUSP1 is a nuclear phosphatase and its major substrates are JNK, p38, and ERK1/2 (Camps et al., 2000). The data showed that the gene expression of DUSP1 was downregulated to 0.64 in the coated dish compared to the non-coated dish (Fig. 1E). This suggested a role for DUSP1 to regulate RANKL expression to mediate osteoclast differentiation. To test this idea, the expression level of
To complement the PCR experiments,
In order to analyze the transcriptional activity of the RANKL gene promoter in response to the knockdown of
To determine the inverse relationship between DUSP1 and RANKL expression, the transcription factor binding sites in the RANKL promoter region were examined using an
The potential association between DUSP1 gene expression and pathologic bone diseases in humans was examined by analyzing publicly available datasets in GEO (accession number GSE12021; Huber et al., 2008). The microarray results were grouped as the young control (n = 5), elderly control (n = 4), rheumatoid arthritis (RA) patients (n = 12), and osteoarthritis (OA) patients (n = 10). In this dataset, DUSP1 mRNA expression in the elderly control was 50% of that of the young control, which implies an aging-dependent decline in the levels of DUSP1 (Fig. 2D). More importantly, in accordance with our results, the DUSP1 expression level was downregulated considerably in RA patients and appeared to be the lowest in OA patients. From this perspective, it is likely that the downregulated levels of DUSP1 allows the enhanced expression of RANKL, which eventually results in the destruction of bone observed in RA and OA patients. Further studies will be needed to examine the precise mechanisms underlying the regulation of osteoclastogenesis.
The results reported here suggest that calcium-containing crystals play a key role in the progression of osteoclastogenesis through the elevated expression of RANKL from osteoblasts. Figure 2E summarizes a possible mechanism of cell-cell signaling that leads to increased osteoclastogenesis. We speculate that the crystals might downregulate DUSP1 gene expression in pre-osteoblasts, which in turn induce RANKL expression via pathways yet to be elucidated. Upregulated RANKL binds to its receptor on pre-osteoclasts, inducing the differentiation of osteoclasts.
This paper reports for the first time using a coculture system that a Ca2+-phosphate coating similar to the crystals found in the articular cartilage of OA patients may stimulate RANKL-mediated osteoclastogenesis. The results provide possible confirmation of the pathogenic role of crystal formation in the development of osteoarthritis. In addition, the present results have shown that the DUSP1 gene is a potential negative regulator of RANKL expression, which appears to be downregulated in both
Mol. Cells 2019; 42(2): 183-188
Published online February 28, 2019 https://doi.org/10.14348/molcells.2018.0382
Copyright © The Korean Society for Molecular and Cellular Biology.
YunJeong Choi1, Ji Hyun Yoo1, Youngkyun Lee2, Moon Kyoung Bae1, and Hyung Joon Kim1,*
1Department of Oral Physiology, BK21 PLUS Project, and Institute of Translational Dental Sciences, School of Dentistry, Pusan National University, Yangsan, Korea, 2Department of Biochemistry, School of Dentistry, Kyungpook National University, Daegu, Korea
Correspondence to:*Correspondence: hjoonkim@pusan.ac.kr
Osteoarthritis (OA) is a naturally occurring, irreversible disorder and a major health burden. The disease is multifactorial, involving both physiological and mechanical processes, but calcium crystals have been associated intimately with its pathogenesis. This study tested the hypothesis that these crystals have a detrimental effect on the differentiation of osteoclasts and bone homeostasis. This study employed an osteoblast-osteoclast coculture system that resembles
Keywords: DUSP1, osteoarthritis, osteoclastogenesis, RANKL
Osteoarthritis (OA) is the most common joint disorder in humans (Fuerst et al., 2009). The disease is slowly progressive and involves all components of the joint, including the bone, cartilage, meniscus, and synovium (Macmullan and McCarthy, 2010). The high prevalence and related physical impairment make OA a leading cause of disability worldwide (McCarthy et al., 2009). Thus far, multiple predisposing factors have been identified, including genetic predisposition, joint trauma, obesity, sex, and hormonal status (Brandt et al., 1998). However, our understanding of the pathogenesis of the condition is limited and there is no pharmacological therapy yet to reverse or retard the consequences of OA (Qvist et al., 2008).
Calcification of the articular cartilage is a well-recognized feature of OA and current evidence suggests that it contributes directly to joint degeneration (MacMullan et al., 2010). Calcium pyrophosphate dehydrate (CPP) and basic calcium phosphate (BCP) are the two most common forms of calcium crystals found in the articular cartilage (Molloy and McCarthy, 2006). The presence of these crystals is associated with a number of clinical manifestations (MacMullan et al., 2010). Both types of crystals are found in OA. Individual crystals are typically less than 1 μm in size and they aggregate in the OA synovial fluid to form clumps that are approximately 5 to 20 μm (McCarthy et al., 2011).
In the present study, we hypothesized that these crystals may have a detrimental effect on the differentiation and survival of osteoblasts and osteoclasts. Recent studies have frequently used the term “coupling of bone and cartilage” to describe the crosstalk between the articular cartilage and subchondral bone in the synovial joints through increased vascularization and development of microcracks in the bone matrix (Burr and Radin, 2003; Lajeunesse and Reboul, 2003) and the cellular and molecular interactions between osteoblasts and chondrocytes that affects the initiation and progression of OA (Karsdal et al., 2008; Mansell et al., 2007; Sharma et al., 2013). Thus far, the pathogenic role of crystals in the degradation of the extracellular matrix and subchondral bone remodeling have been studied (Corr et al., 2017) but only few studies have examined their effects on osteoclasts and bone remodeling (Stack and McCarthy, 2016).
To test this hypothesis, amorphous calcium phosphate (ACP) arrays were first prepared on a 60 mm culture dish, as described previously (Kim et al., 2010). ACP arrays were fabricated on the surface of the dish to mimic the crystals observed in OA patients. As illustrated in Fig. 1(A), murine bone marrow-derived macrophages (BMMs) were cocultured with murine calvarial osteoblasts, as described by Ryu et al. (Ryu et al., 2006) either with or without a Ca2+-phosphate coating. Early studies of cocultures found that a direct cell-to-cell interaction was required for osteoclast generation, while the following studies showed that RANKL expressed on osteoblasts and RANK present on the osteoclast precursors were responsible for the cell surface molecular interaction for osteoclastic differentiation (Suda et al., 1999). The cocultures were treated with prostaglandin E2 (10−6 M) and vitamin D3 (10−8 M) to stimulate osteoblast differentiation and induce cell surface RANKL expression to facilitate the osteoclastic differentiation of BMMs, which resemble the actual osteoblast-dependent osteoclast differentiation
In addition, gene expression of the common markers of osteoclast activity, cathepsin K, and matrix metallopeptidase 9 (MMP9) was assessed (Fig. 1C). In keeping with the earlier results, it was found that the expression of those two genes were increased notably in osteoclastic cells cultured in the coated dish on day 6. In an attempt to identify the molecular mechanisms underlying the augmentation of osteoclastogenesis by the crystals in the coculture, the altered expression of genes between the non-coated and coated conditions were analyzed using MouseWG-6 v2.0 Expression BeadChip (Illumina). Microarray results from Fig. 1D revealed that the gene expression of some common markers of osteoblastic activity, such as collagen type 1, bone sialoprotein, and Runx2, did not show a significant difference between the two dishes on day 6 of coculture. Conversely, gene expression of RANKL, which plays an essential role in the commitment of precursors to osteoclastic differentiation (Boyle et al., 2003; Suda et al., 1999; Teitelbaum and Ross, 2003), was upregulated by 140% from 237.1 to 326.9, suggesting that a Ca2+-phosphate coating does not significantly alter osteoblast differentiation but enhances osteoclast differentiation.
The microarray results from cultures of osteoblasts grown on either non-coated or coated dishes for six days were next examined to determine the gene responsible for the increase in RANKL expression. Microarray analysis identified the genes that showed a greater than 1.35-fold difference in expression between the two dishes. Among the 167 genes, two genes reported the greatest difference and dual-specificity phosphatases 1 (DUSP1) was selected as the most relevant after pathway analysis using PANTHER (Mi et al., 2013). DUSPs are cysteine-based enzymes that can remove phosphate groups from phosphor-serine/threonine residues (Patterson et al., 2009) and play important roles in MAPK signaling pathway in the development and immune response (Nunes-Xavier et al., 2011). Among them, DUSP1 is a nuclear phosphatase and its major substrates are JNK, p38, and ERK1/2 (Camps et al., 2000). The data showed that the gene expression of DUSP1 was downregulated to 0.64 in the coated dish compared to the non-coated dish (Fig. 1E). This suggested a role for DUSP1 to regulate RANKL expression to mediate osteoclast differentiation. To test this idea, the expression level of
To complement the PCR experiments,
In order to analyze the transcriptional activity of the RANKL gene promoter in response to the knockdown of
To determine the inverse relationship between DUSP1 and RANKL expression, the transcription factor binding sites in the RANKL promoter region were examined using an
The potential association between DUSP1 gene expression and pathologic bone diseases in humans was examined by analyzing publicly available datasets in GEO (accession number GSE12021; Huber et al., 2008). The microarray results were grouped as the young control (n = 5), elderly control (n = 4), rheumatoid arthritis (RA) patients (n = 12), and osteoarthritis (OA) patients (n = 10). In this dataset, DUSP1 mRNA expression in the elderly control was 50% of that of the young control, which implies an aging-dependent decline in the levels of DUSP1 (Fig. 2D). More importantly, in accordance with our results, the DUSP1 expression level was downregulated considerably in RA patients and appeared to be the lowest in OA patients. From this perspective, it is likely that the downregulated levels of DUSP1 allows the enhanced expression of RANKL, which eventually results in the destruction of bone observed in RA and OA patients. Further studies will be needed to examine the precise mechanisms underlying the regulation of osteoclastogenesis.
The results reported here suggest that calcium-containing crystals play a key role in the progression of osteoclastogenesis through the elevated expression of RANKL from osteoblasts. Figure 2E summarizes a possible mechanism of cell-cell signaling that leads to increased osteoclastogenesis. We speculate that the crystals might downregulate DUSP1 gene expression in pre-osteoblasts, which in turn induce RANKL expression via pathways yet to be elucidated. Upregulated RANKL binds to its receptor on pre-osteoclasts, inducing the differentiation of osteoclasts.
This paper reports for the first time using a coculture system that a Ca2+-phosphate coating similar to the crystals found in the articular cartilage of OA patients may stimulate RANKL-mediated osteoclastogenesis. The results provide possible confirmation of the pathogenic role of crystal formation in the development of osteoarthritis. In addition, the present results have shown that the DUSP1 gene is a potential negative regulator of RANKL expression, which appears to be downregulated in both
Bo Hyun Kim, Ju Hee Oh, and Na Kyung Lee
Mol. Cells 2017; 40(10): 752-760 https://doi.org/10.14348/molcells.2017.0098Seung Ah Lee, Jin Hee Park, and Soo Young Lee
Mol. Cells 2013; 36(3): 273-277 https://doi.org/10.1007/s10059-013-0226-3Jeong-Yeon Seo, Tae-Hyeon Kim, Kyeong-Rok Kang, HyangI Lim, Moon-Chang Choi, Do Kyung Kim, Hong Sung Chun, Heung-Joong Kim, Sun-Kyoung Yu, and Jae-Sung Kim
Mol. Cells 2023; 46(4): 245-255 https://doi.org/10.14348/molcells.2023.2149